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Reports a study on the glassy magnetic properties of the amorphous phase of the magnet Co[sub3]BTCA[sub2](H[sub2]O)[sub6]. Experiment conducted; Results; Discussion.

Thromboxane A2 (TXA2) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA2 receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro- and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA2 synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro- and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues. [ABSTRACT FROM AUTHOR]

The thromboxane A(2) receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. We have previously shown that TPbeta, but not TPalpha, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPbeta, but not TPalpha, also undergoes tonic internalization. Tonic internalization of TPbeta was temperature- and dynamin-dependent and was inhibited by sucrose and NH(4)Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX(3)phi motif (where X is any residue and phi is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPbeta was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX(2)phi or YX(3)phi motif in the carboxyl-terminal tail of TPalpha induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPbeta undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPbeta. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPbeta and represent the first report of a YX(3)phi motif involved in tonic internalization of a cell surface receptor.

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is considered to play a critical role in the desensitization of responses mediated by these receptors. To explore the role of GRK2 in A2 adenosine receptor desensitization, we attempted to reduce specifically GRK2 expression in NG108-15 cells by stable transfection with an antisense rat GRK2 cDNA sequence. This yielded up to a 69% loss of GRK2 when compared with plasmid-transfected control cells, which correlated with a reduction in kinase activity when measured by the ability of cell lysates to promote light-dependent phosphorylation of rhodopsin. Levels of GRK3 were the same in antisense and plasmid-transfected controls. On addition of the A2 adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was greater in GRK2 antisense cells as compared with plasmid control cells. In contrast, cyclic AMP accumulation via agonist stimulation of either IP-prostanoid or secretin receptors or by addition of forskolin was not significantly different among all clones examined. The increase in A2 adenosine receptor response could not be explained by changes in A2A adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-3H-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl]phenol ([3H]ZM241385). These data show for the first time a direct correlation between expression of GRK2 and desensitization of natively expressed A2 adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors. Key Words: G protein-coupled receptor kinase-G protein-coupled receptor-Antisense-NG108-15 cells-A2 adenosine receptors-Desensitization.

Beta-agonists, through activation of the beta(2)-adrenergic receptor (beta(2)AR)-G(s)-adenylyl cyclase (AC) pathway, promote bronchodilation via functional antagonism of airway smooth muscle (ASM) spasmogens associated with the asthmatic state. Although previous studies have demonstrated that beta(2)AR signaling in ASM is subject to homologous (beta-agonist-induced) beta(2)AR desensitization, the potential for inflammatory and contractile agents to impact beta(2)AR signaling in ASM through heterologous mechanisms has not been defined. Here we report that chronic exposure of human ASM (HASM) to carbachol, serotonin, the thromboxane analogue U46619, or histamine induced little change or a small increase in isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) formation, but significantly increased cAMP formation elicited by stimulation with forskolin. This latter increase in intrinsic AC activity was largely reversed by pertussis toxin pretreatment, and was unaffected by protein kinase C inhibition. Analysis of both AC function and isoform expression supports a dominant role of AC VI in HASM, and points to important differences in ASM AC isoform expression among species. Additional studies identify AC as the limiting component in beta(2)AR-G(s)-AC signaling in HASM, and thus a potentially important target of therapeutic strategies designed to influence airway contractile state.

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two-to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory process through modulation of chemotactic receptor expression.

The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is known regarding the usefulness of these compounds as inhibitors in intact cells. In attempting to characterize the role of protein kinase A (PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an effective inhibitor of intracellular PKA, extended analysis of the compound determined the principal effect of H-89 was via its action as a beta-2 AR antagonist. Pretreatment with or the acute addition of H-89 significantly attenuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response to isoproterenol suggested beta-2 AR antagonism by retained H-89. Competition binding of [125I]iodopindolol established Ki values of approximately 180 nM and 350 nM for H-89 antagonism of beta-2 AR and beta-1 AR, respectively. Additional receptor binding studies suggest selectivity of H-89 for the beta-2 AR and beta-1 AR, although a weak antagonism (Ki values of approximately 10 microM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological inhibitors when used in intact cell models.

Platelet-activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys90, Cys95 and Cys173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-alpha (100-400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-alpha-pretreated monocytes. These observations provide new evidence for TNF-alpha and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-alpha.

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.

Platelet-activating factor is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor (GPCR) family. In the present report, we demonstrate that the highly conserved aspartate 63 is critical in G protein coupling of the PAF receptor: substitution of an asparagine for the aspartate 63 (D63N) abolished inositol phosphate production following agonist stimulation; moreover, binding isotherms of the D63N mutant were monophasic and unaffected by GTPgammaS. We also demonstrate that aspartate 63 is not involved in direct interaction with the agonist: the D63N mutant displayed a higher intrinsic affinity for PAF than the uncoupled WT receptor. Sodium decreased specific (3)H-PAF and antagonist (3)H-WEB2086 binding to the PAF receptor, but the aspartate 63 residue was not involved in this regulation, contrary to cognate aspartate residues in other GPCRs. Our data suggest that aspartate 63 in the PAF receptor may be involved in the structural requirement for G protein coupling to the receptor and may contribute to receptor affinity for the ligand.

Platelet-activating factor is a potent phospholipid mediator which produces a wide range of biological responses by binding to a specific, high-affinity receptor on the target cell surface. Platelet-activating factor has been shown to have a very important pro-inflammatory role while the cytokine transforming growth factor-beta, also known for its diverse and potent effects, can act as both a pro- and anti-inflammatory agent. To ascertain whether transforming growth factor-beta could modulate the expression of the human platelet-activating factor receptor, different cell types were stimulated with this cytokine and the effect determined by Northern blot analysis and Ca2+ influx studies. The results demonstrate that transforming growth factor-beta induces the expression of the platelet-activating factor receptor in monocytic and B cell lines and indicate that transforming growth factor-beta could play a role in the modulation of the inflammatory response by up-regulating the expression of the platelet-activating factor receptor.